1. Field of the Invention
The present invention relates to the field of melanocyte cell lines.
2. Summary of the Invention
Melanoma is a malignant neoplasm derived from cells capable of forming melanin. Melanoma, together with squamous cell and basal cell carcinoma, comprises skin cancer, the most commonly diagnosed cancer in the U.S. Melanoma represents a serious public health problem. It is the fifth most common cancer in the United States, and is associated with the highest case fatality rate of all skin cancers. (Rigel, D. S. Malignant Melanoma: perspectives in incidence and its effects on awareness, diagnosis and treatment, CA Cancer J. Clin. 1996 46: 195-198; MMWR May 3, 1996 45, #17).
Melanocytes and melanoma cells express melanogenisis-related proteins (Orlow et al. 1995; del Marmol and Beerman, 1996). These proteins contribute to the antigenicity of melanomas, making them attractive targets for cancer vaccines.
Hgp100 is a melanoma-associated glycoprotein antigen that is closely related to the melanogenesis-related protein pmel17. These proteins differ at the genetic level by an in frame deletion of a 21 base pair sequence in hgp100 (Adema et al. 1994). For purposes of this disclosure, gp100 and pmel17 are used interchangeably.
gp100 is an attractive candidate for a cancer vaccine against melanoma. Prototype vaccines based on synthetic gp100 peptides or recombinant viral vectors are currently in phase I clinical trials. Studies on gp100 polypeptide have been hampered by the limited availability of purified native or recombinant protein. It is difficult and impractical to purify hgp100 (human gp100) from melanoma specimens, and such approaches do not yield sufficient material for use in vaccines.
Recombinant technology should provide high quality, highly purified hgp100 in sufficient quantity to be useful in therapeutic cancer vaccines. To date, only one group has reported success in cloning and expressing of hgp100 in vitro. (Huang et al. J. Invest Dermatol.) Huang et al. derived the hgp100 gene from an established melanoma cell line and cloned the gene into E. coli. However, hgp100 is a glycoprotein and prokaryotes are not capable of glycosylating recombinant proteins. Thus, expression of cloned hgp100 in E. coli leads to the production of a recombinant product that differs in significant respects from the native protein. Critical antigenic determinants may be lost, limiting the applicability of such proteins to vaccines. Thus, a need exists for a method of production of high levels of glycosylated hgp100 Ag in a form more closely related to that which exists in nature.
The present invention provides cells, designated WC-1 14.07 (ATCC accession no. PTA-1275), that can be used as a source of hgp100. Cells of the WC-1 14.07 eukaryotic cell line endogenously express hgp100, and since hgp100 is a known tumor-associated antigen, cells of the WC-1 14.07 cell line are a useful source of hgp100 for use in immunological formulations, e.g., as a vaccine to treat patients with melanoma. Eukaryotic cell line WC-1 14.07 is also a useful source of hgp100 for use as a research reagent for immunologists studying the cell-mediated immune responses to hgp100 and biological processes involving hgp100. The cells can be inactivated and used directly to stimulate the host immune response to hgp100. Alternatively, hgp100 can be extracted and purified from the cells using affinity chromatography (Wilchek, M., Miron, T., and Kohn J. 1984. Affinity Chromatography. Methods Enzymol. 104: 3-55). hgp100 could be used in immunological assays to measure T cell proliferation or antibodies to hgp100.
The cells of the present invention can also be used as a source of melanin. Melanin is produced in melanosomes and is transferred to keratinocytes. It has been shown to reduce the amount of chromosome aberrations when injected directly into cells prior to radiation exposure (Mosse, I, Kostrova L, Subbot S, Maksimenya I, Molophei V.) Melanin decreases clastogenic effects of ionizing radiation in human and mouse somatic cells and modifies the radioadaptive response. Radiat Environ Biophys 2000 Mar; 39(1):47-52). Melanin can also be used for the therapeutic and cosmetic modification of hair. Melanin targeted to hair follicles by topical application results in the restoration of hair pigment (Hoffinan RM. Topical liposome targeting of dyes, melanins, genes and proteins selectively to hair follicles. J Drug Target 1998;5(2):67-74).
The cells of the present invention can also be used as a source of s100. s100 is a multigenic family of Ca2+ binding proteins and has 19 different members that are differentially expressed in a large number of cell types. Members of s100 have been implicated in a number of Ca2xe2x88x92 dependent activities including protein phosphorylation, enzyme activities, cell proliferation and neoplastic transformation and differentiation, cytoskeleton, membrane organization, inflammation and oxidative cell damage (Donato R. Functional roles of s100 proteins, calcium-binding proteins of the EF-hand type. Biochim Biophys Acta 1999 Jul 8:1450(3):191-231). Altered expression of s100 proteins is associated with several human disorders including cancer, neurodegenerative diseases, cardiomyopathies, inflammations, diabetes and allergies (Heizmann CW, Cox JA. New perspectives on s100 proteins: a multi-functional Ca(2+)-, Zn(2+)- and Cu(2+)-binding protein family. Biometals 1998 Dec;11(4):383-97). Thus, s100 could be prepared from cells of the present invention and used in the research and treatment of these disorders.
Yet another use of the cells of the present invention is as a universal recipient for expression of hgp100 in the context of pre-determined Class I MHC determinants. Since WC-1 14.07 has lost its MHC Class I expression, transfection of this cell line with plasmids encoding any MHC Class I molecule results in expression of cell surface MHC Class I molecules with appropriate hgp100 peptides. These cells, modified by transfection, comprise yet another aspect of the present invention. Such cells can readily be prepared by standard molecular biology methods, see, e.g., Maniatis et al., and be used, for example, as targets for lysis by cytotoxic T-lymphocytes. These transfected cells could eliminate the need for an autologous cell line when monitoring patients for specific hgp100 epitopes. One target cell line could be used for all patients being monitored during a clinical trial.
The practice of the present invention will employ, unless otherwise indicated, conventional methods of immunology, molecular biology, cell biology and recombinant DNA techniques known to those skilled in the, art. Such techniques are explained fully in the literature. See, e.g., Sambrook, et al., MOLECULAR CLONING, A LABORATORY MANUAL, Second Ed. (1989); Nucleic Acid Hybridization (B. D. Hames and S. J. Higgins, 1984); Animal Cell Culture (R. Freshney ed. 1986); Immunochemical Methods in Cell and Molecular Biology (Mayer and Walker, eds., Academic press, London, 1987); Protein Purification: Principles and Practice, Second Edition (Scopes, ed., Springer-Verlag, N.Y. 1987); Current Protocols in Immunology (John Wiley and Sons, NY 1998); Antibodies, a Laboratory Manual (Ed Harlow and David Lane, eds, Cold Spring Harbor, N.Y. 1988); and Fundamental Immunology (Paul, ed, Raven Press, NY 1993).
The present invention provides a stable, adherent, continuous melanocyte cell line, designated WC-1 14.07 (ATCC accession no. PTA-1275). The cell line was obtained by culturing the peripheral blood mononuclear cells of an adult male human volunteer under conditions that allowed for the selection of stable adherent cells. The WC-1 14.07 line, expanded from cells selected after 60 days of weekly subculture in standard tissue culture medium, is of melanocyte origin. Characterization of WC-1 14.07 revealed the following phenotype: s100, hgp100 and melanin positive; CD11c, CD80, CD86 negative; weakly CD14+; HLA DR, DQ, DP negative; and MHC Class I/II negative.
The melanocyte cell line of the present invention provides a useful source of hgp100, melanin and s100. The stability and continuous nature of this cell line makes it amenable to large scale culture in tissue culture. Cells are then isolated, disrupted by standard techniques, and constituents of interest purified by standard techniques known to those skilled in the art. For example, immunoaffinity columns are prepared by immobilizing antibody to hgp100 on cyanogen boride-activated Sepharose 4B. Extracts of the melanocyte cell line WC-1 14.07 are then passed over the immunoaffinity column under conditions that allow for antigen-antibody binding. Non-specific cell constituents and debris pass through the immunoaffinity column and are discarded or used for other purposes. The hgp100 is then eluted from the immunoaffinity column by washing with a buffer that disrupts the antigen-antibody bond (e.g., by applying a pH or salt gradient). Similarly, antibodies to melanin and s100 can be used to prepare specific immunoaffinity columns with which to purify melanin or s100 from extracts of WC-1 14.07.
The cells of the present invention can also be modified by transfection to express heterologous proteins, such as, in particular, predetermined MHC constituents. Transfection with a class I histocompatibility gene under conditions favorable to expression of such gene then provides a modified melanocyte cell line that produces hgp100 (or other constituents) in the context of the predetermined MHC molecule. Thus, hgp100 is presented on the surface of such modified cell together with the MUC determinant and will thereby be efficiently presented to T-cells. Standard methods well known to those skilled in the art can be employed to effect transfection.